Sensitive detection of human cytomegalovirus peptide–specific cytotoxic T-lymphocyte responses by interferon-γ–enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation

Reconstitution of human cytomegalovirus (HCMV)–specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensiti...

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Veröffentlicht in:Blood 2002-05, Vol.99 (10), p.3830-3837
Hauptverfasser: Hebart, Holger, Daginik, Senay, Stevanovic, Stefan, Grigoleit, Ulrich, Dobler, Andrea, Baur, Manuela, Rauser, Georg, Sinzger, Christian, Jahn, Gerhard, Loeffler, Juergen, Kanz, Lothar, Rammensee, Hans-Georg, Einsele, Hermann
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container_end_page 3837
container_issue 10
container_start_page 3830
container_title Blood
container_volume 99
creator Hebart, Holger
Daginik, Senay
Stevanovic, Stefan
Grigoleit, Ulrich
Dobler, Andrea
Baur, Manuela
Rauser, Georg
Sinzger, Christian
Jahn, Gerhard
Loeffler, Juergen
Kanz, Lothar
Rammensee, Hans-Georg
Einsele, Hermann
description Reconstitution of human cytomegalovirus (HCMV)–specific cytotoxic T lymphocytes (CTLs), predominantly directed against pp65, provides protective immunity for the development of HCMV disease after allogeneic stem cell transplantation (SCT). To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-γ release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8+ T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide–specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen–specific CD4+ T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35->180 days). Peak counts of peptide-specific CD8+ T cells ranged from 0.14 to 60.6 cells/μL; those of protein-specific CD4+ T cells ranged from 0.64 to 18.97 cells/μL. Reconstitution of HCMV-peptide–specific CD8+ T cells and protein-specific CD4+ T cells was associated with clearance of HCMV infection (r2 = 0.89, P < .0001 and r2 = 0.61, P = .0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein–specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4+ and CD8+ T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.
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To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-γ release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8+ T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide–specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen–specific CD4+ T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35-&gt;180 days). Peak counts of peptide-specific CD8+ T cells ranged from 0.14 to 60.6 cells/μL; those of protein-specific CD4+ T cells ranged from 0.64 to 18.97 cells/μL. Reconstitution of HCMV-peptide–specific CD8+ T cells and protein-specific CD4+ T cells was associated with clearance of HCMV infection (r2 = 0.89, P &lt; .0001 and r2 = 0.61, P = .0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein–specific T cells at 3 months after SCT. 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To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-γ release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8+ T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide–specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen–specific CD4+ T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35-&gt;180 days). Peak counts of peptide-specific CD8+ T cells ranged from 0.14 to 60.6 cells/μL; those of protein-specific CD4+ T cells ranged from 0.64 to 18.97 cells/μL. Reconstitution of HCMV-peptide–specific CD8+ T cells and protein-specific CD4+ T cells was associated with clearance of HCMV infection (r2 = 0.89, P &lt; .0001 and r2 = 0.61, P = .0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein–specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4+ and CD8+ T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.</description><subject>Adult</subject><subject>Aged</subject><subject>Anesthesia. Intensive care medicine. Transfusions. 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To define pp65-derived CTL epitopes that would allow sensitive detection of HCMV-specific immune reconstitution, a computer-based epitope prediction was performed. Peptide-specific CTL responses were assessed by interferon-γ release. With this approach, pp65-derived epitopes presented by the HLA alleles A*0101, A*0201, A*1101, and B*0702 were identified. The frequency of CTLs in healthy HCMV-seropositive individuals ranged from about 0.1% to 3.3% of all CD8+ T cells. In patients at risk of HCMV infection after allogeneic SCT, HCMV-peptide–specific CTLs were found in 14 of 19 patients at a median of 90 days after SCT (range, 35-234 days) and HCMV-antigen–specific CD4+ T lymphocytes in 11 of 18 patients at a median of 90 days after SCT (range, 35-&gt;180 days). Peak counts of peptide-specific CD8+ T cells ranged from 0.14 to 60.6 cells/μL; those of protein-specific CD4+ T cells ranged from 0.64 to 18.97 cells/μL. Reconstitution of HCMV-peptide–specific CD8+ T cells and protein-specific CD4+ T cells was associated with clearance of HCMV infection (r2 = 0.89, P &lt; .0001 and r2 = 0.61, P = .0045, respectively). HCMV infection recurred after documentation of HCMV-specific T-cell reconstitution (n = 4) when immunosuppression was intensified. Patients in whom late-onset HCMV disease developed lacked HCMV-protein–specific T cells at 3 months after SCT. In conclusion, prospective monitoring of HCMV-specific CD4+ and CD8+ T-cell reconstitution can be performed rapidly by using flow cytometry after specific stimulation with HCMV peptides and proteins and might help to further improve clinical management of HCMV infection after allogeneic SCT.</abstract><cop>Washington, DC</cop><pub>Elsevier Inc</pub><pmid>11986243</pmid><doi>10.1182/blood.V99.10.3830</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record>
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subjects Adult
Aged
Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy
Antibodies, Viral - blood
Antigens, Viral - immunology
Biological and medical sciences
Blood Donors
Bone marrow, stem cells transplantation. Graft versus host reaction
Cell Line
Cells, Cultured
Cytomegalovirus - immunology
Cytomegalovirus Infections - immunology
Cytomegalovirus Infections - prevention & control
Enzyme-Linked Immunosorbent Assay - methods
Epitopes - immunology
Female
Flow Cytometry - methods
Hematopoietic Stem Cell Transplantation - adverse effects
Hematopoietic Stem Cell Transplantation - methods
Humans
Interferon-gamma - analysis
Kinetics
Leukapheresis
Male
Mathematical Computing
Medical sciences
Middle Aged
Peptides - immunology
Phosphoproteins - immunology
Sensitivity and Specificity
T-Lymphocytes, Cytotoxic - immunology
Transfusions. Complications. Transfusion reactions. Cell and gene therapy
Transplantation, Homologous
Viral Matrix Proteins - immunology
title Sensitive detection of human cytomegalovirus peptide–specific cytotoxic T-lymphocyte responses by interferon-γ–enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation
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